Morphological and ecological convergence at the lower size limit for vertebrates highlighted by five new miniaturised microhylid frog species from three different Madagascan genera Author Scherz, Mark D. Author Hutter, Carl R. Author Rakotoarison, Andolalao Author Riemann, Jana C. Author Rödel, Mark-Oliver Author Ndriantsoa, Serge H. Author Glos, Julian Author Roberts, Sam Hyde Author Crottini, Angelica Author Vences, Miguel Author Glaw, Frank text PLoS ONE 2019 2019-03-27 213314 1 45 journal article 27810 10.1371/journal.pone.0213314 c3128d24-5293-4125-bc85-0678753fc483 PMC6436692 30917162 2614083 91E597C8-7A80-46F9-B0E8-61F2524400F7 Mini ature sp. nov. urn:lsid:zoobank.org:act: 0C1C4CE2-419A-4030-86D1-B1B89793697D ( Figs 1–3 and 9 , Table 1 ) Remark. This species was previously listed as Stumpffia sp. Ca53 MV2017(MF867231) by Tu et al. [ 20 ], though with incorrect accession number (correct number is MF768231 ). Holotype ( Figs 2 and 9 ). ZSM 86 /2004 ( FGZC 0 151, GenBank accession numbers MF768231 and MK 459307 for 5’ and 3’ fragments of the 16S rRNA gene, respectively, and MF768147 for cox1 gene), a presumed adult specimen collected in Andohahela National Park between Isaka and Eminiminy above ‘ Camp 1’ (ca. 24.75˚S, 46.85˚E, ca. 350 m ) between 29 and 31 January 2004 by F. Glaw , M. Puente , M. Teschke (née Thomas), and R.D. Randrianiaina. Diagnosis. A highly miniaturised frog assigned to Mini gen. nov. on the basis of its small size, curved clavicles, laterally displaced and reduced nasals, and fusion or loss of carpal 2. This assignment is supported by its genetic affinities ( Fig 1 ; [ 20 ]). It is separated by uncorrected pdistances of 10.0–10.6% in the analysed 3’ fragment of the 16S rRNA gene from other members of the genus Mini gen. nov. , and 10.8–13.7% from all members of the genus Plethodontohyla . Mini ature sp. nov. is characterised by the unique combination of the following characters (n = 1 specimen ): (1) SVL 14.9 mm ; (2) ED/HL 0.38; (3) HW/SVL 37.2; (4) FARL/SVL 0.32; (5) TIBL/SVL 0.34; (6) HIL/SVL 1.18; (7) finger 1 strongly reduced, 2 and 4 reduced; (8) toe 1 absent, toes 2 and 5 quite reduced; (9) maxillary and premaxillary teeth present; (10) vomerine teeth absent; (11) lateral colour border absent; (12) black inguinal spots present; (13) postchoanal vomer present, spatulate, medially fused to parasphenoid; (14) nasal cultriform and laterally displaced; (15) quadratojugal-maxilla contact strong and broad; (16) zygomatic ramus of squamosal long, thin, curved, and horizontal; (17) clavicles present, curving with simple lateral articulations, medially not bulbous; (18) prepollex thin and cultriform; (19) carpal 2 absent or fused to post-axial carpal 3–5 element; (20) finger phalangeal formula 1-2-3-3; (21) toe phalangeal formula 1-2-3-4-3; (22–26) calls unknown. Within the genus Mini gen. nov. , the new species can be distinguished by its distinctly larger body size ( 14.9 mm vs 8.2–11.3 mm ), shorter relative hindlimb length (HIL/SVL 1.18 vs 1.41–1.72) and phalangeal formula of fingers (1-2-3-3 vs 1-2-3- 2 in M . mum sp. nov. and 0-2- 3- 2 in M . scule sp. nov. ). Additionally, it can be distinguished from M . mum sp. nov. by the presence of maxillary and premaxillary teeth (vs absence), and less distinct lateral colour Fig 9. Osteology of Mini ature gen. et sp. nov. holotype (ZSM 86/2004). (a-c) Whole skeleton in (a) dorsal, (b) lateral, and (c) ventral view. (d-g) Skull in (d) lateral, (e) ventral, (f) anterior, and (g) dorsal view. (h) Foot in ventral view. (i) Hand in ventral view. For abbreviations, see Fig 6. https://doi.org/10.1371/journal.pone.0213314.g00 9 border, and M . scule sp. nov. by proportionally smaller nasals and braincase, broader quadratojugal-maxillary contact, and vertical dorsal process of premaxilla (vs anterior). This species is particularly similar to some highly miniaturised Stumpffia species, but it can be distinguished from all Stumpffia based on the condition of the carpals and the presence of curved clavicles, and most Stumpffia by the presence of maxillary and premaxillary teeth. Holotype description. Specimen in a moderately good state of preservation, the left arm removed as a tissue sample, the whole body somewhat dorsoventrally flattened in preservative. Body oblong; head wider than long, narrower than body width; snout slightly pointed in dorsal view, pointed in lateral view; nostrils directed laterally, not protuberant, equidistant between tip of snout and eye; canthus rostralis rounded, indistinct, slightly concave; loreal region flat, vertical; tympanum indistinct, round, ~48% of eye diameter; supratympanic fold absent; tongue long, broadening posteriorly, attached anteriorly, not notched; maxillary teeth present; vomerine teeth present; choanae small and round. Forelimb slender; subarticular tubercles single, elongated; outer/palmar metacarpal tubercle small and round; inner metacarpal tubercle slightly smaller than outer/palmar; hand without webbing; first finger strongly reduced, second and fourth fingers reduced, third finger basally broadened; relative lengths of fingers 1 < 2 = 4 < 3, fourth and second finger equal in length; finger tips not expanded into discs. Hindlimbs stocky; TIBL 34% of SVL; lateral metatarsalia strongly connected; inner metatarsal tubercle indistinguishable from first toe; outer metatarsal tubercle absent; no webbing between toes, second and fifth toes reduced; relative lengths of toes 2 < 5 < 3 < 4, fifth toe distinctly shorter than third; toe tips slightly pointed distally. Skin on dorsum smooth without a distinct dorsolateral fold, but with a distinct colour border, see below. Ventral skin smooth. After 14 years in 70% ethanol, the dorsum is light brown, paler—almost beige—laterally, and slightly translucent, with a thin beige vertebral stripe and a darkened area on the posterior head. The skin above the eyes is translucent and dark in colour through the presence of the eyes beneath. The dorsal forelimb is beige flecked with brown, the hand is lighter medially, and the fingers have faint cream annuli. The dorsal hindlimb is beige in base colour with several brown crossbands on the thigh and shank that line up when the leg is folded together. A trapezoid of brown is present around the vent. The foot is dorsally as the forelimb with a light annulus before each distal phalanx. A distinct colour border that is not straight is present laterally, running along the canthus rostralis from the nostril through the eye, through the supratympanic region along the torso to the inguinal region. Small oblong dark brown spots are present in the inguinal region. The side of the head is dark brown. Ventral to this colour bor- der the frog is mocha speckled with beige, lightening ventrally to beige with loose cream speckles. The ventral skin is translucent, and some of the organs can be seen through it. The chin is not differently coloured than the rest of the ventral body. This pattern continues onto the ventral limbs. No data on life colouration are available. Variation. This species is currently known from a single specimen only. Osteology ( Fig 9 ). Based on ZSM 86/2004 (figured). Cranium ( Fig 9 D–9G). Shape and proportions. Skull almost equilateral, roughly as wide as long, widest at quadratojugal-squamosal junction, lateral to the otic region. Braincase moderately broad, rostrum not shortened. Neurocranium. Well ossified, including the otic capsules. The anterior cone of the sphenethmoid is ossified and contacts the frontoparietal dorsally but is not in contact with any other bones; small isolated lateral mineralisations of this bone are also present anterodorsal to the anterior tip of the cultriform process of the parasphenoid. Prootic in dorsal contact with lateral flange of frontoparietal, ventral contact with parasphenoid alae, not approaching contralateral ventrally. Septomaxilla miniscule, relatively poorly mineralised, and therefore not further discussed here. Columella (stapes) well ossified, pars media plectra (stylus) long and nearly straight, weakly posteriorly and dorsally oriented toward the reniform, dorsally elongated pars interna plectra (baseplate). Nasal narrow and cultriform, laterally displaced, curved downward laterally, acuminate maxillary process not closely approaching maxillary pars facialis, broadly separated from contralateral. Frontoparietal with rounded anterior edge, laterally bulging, with short lateral flange covering prootic, posteriorly strongly connected to exoccipital, anteroventrally contacting sphenethmoid, lacking any dorsal process. Parasphenoid with narrow, rather straight-edged cultriform process and broad perpendicular alae, considerably shorter than frontoparietals, in contact with exoccipitals posterodorsally, prootics dorsally along the edges of the alae, anteroventrally in contact with postchoanal vomer and not in contact with neopalatine; posteromedial process excluded from participating in foramen magnum by exoccipitals. Vomer divided into pre- and postchoanal portions; prechoanal portion narrow, arcuate, triradiate, with a short lateral and anterior ramus and long, curving posterior ramus; postchoanal portion spatulate and edentate, contacting its contralateral on the midline, in dorsal contact with the parasphenoid proximally and the neopalatine distally, lacking an anterior projection. Neopalatine simple, straight, almost indistinguishable from lateral postchoanal portion of vomer, laterally broadly separated from the maxilla, not exceeding the lateral-most point of the postchoanal vomer. Maxillary arcade fairly slight, maxilla and premaxilla bearing numerous diminutive teeth, anterior extension of maxilla exceeding lateral extent of premaxilla but not in contact with it. Premaxilla with a broad acuminate dorsal alary process rising laterally, pars palatina shallowly divided into a narrow palatine process and broad lateral process. Maxilla with a low triangular pars facialis and a narrow pars palatina, its posterior tip acuminate and broadly overlapping the quadratojugal, the lingual surface of the pars palatina running parallel to but not touching the anterior ramus of the pterygoid. Pterygoid with a short medial ramus, long anterior ramus, and broad posterior ramus and posterolaterally calcified to the quadratojugal complex. Quadratojugal weakly bowed laterally, broadly connected to the ventral ramus of the squamosal, bearing a small posteroventral knob; the articulation of the mandible is apparently fortified by the mineralisation of the posterior ramus of the pterygoid+squamosal+quadratojugal posteroventral knob. Squamosal with a slender ventral ramus, broadened otic ramus, and long, thin zygomatic ramus, the otic ramus oriented slightly dorsally and posteriorly, the zygomatic ramus horizontal and strongly curved. Mandible slim and edentate, largely unremarkable, with a low coronoid process on the angulosplenial. Mentomeckelians separated from the dentary, with slightly bulbous, almost hooked ventrolateral projections. Posteromedial processes of hyoid proximally rounded with a broad medial crista. Postcranial skeleton ( Fig 9 A–9C, 9H and 9I ). Eight procoelous unfused presacrals, much broader than long, lacking neural spines, with round posterior articular processes, presacral I with a complete neural arch, presacrals II–IV with thicker and longer transverse processes than V–VIII. Sacrum with expanded diapophyses, the leading and trailing edges roughly equally angled, the articulation type IIB sensu Emerson [ 42 ]. Urostyle bicondylar, long, not broadening posteriorly, without lateral processes and with a low dorsal ridge. Pectoral girdle without ossified prezonal or postzonal elements, with ossified clavicles. Clavicle thin and curved, with a simple lateral junction, shorter than the coracoid. Coracoid fairly narrow, not flared laterally, strongly flared medially with a large medial articular surface with the contralateral. Scapula slender, with a thin pars acromialis, the cleithral border concave. Cleithrum ossified for two thirds the width of the scapular border, thickened anteriorly. Suprascapula unossified. Humerus with a well-developed crista ventralis and no medial or lateral cristae. Radioulna slender with a distinct sulcus intermedius. Carpals poorly ossified, composed of radiale, ulnare, prepollical element, element Y, and large post-axial element formed by carpals 3–5. Carpal 2 has either been lost or fused to the latter element. Finger phalangeal formula is reduced (1-2-3- 3), and the terminal phalanx of the first finger is a small, round element. Small distal knobs on terminal phalanx of finger 3. Prepollex thin and cultriform and extending only to the base of the first metacarpal. Pubis calcified, iliac shafts passing ventral to and beyond sacrum, nearly cylindrical, without a dorsal crest and with a weak dorsal prominence and shallow oblique groove. Femur weakly sigmoid, almost lacking a posterior crest. Tibiofibula shorter than femur, with a sulcus intermedius. Tibiale and fibulare fused proximally and distally. T1 and T2+3 tarsals present, T1 considerably smaller than T2+3, plus a small additional ossification (possibly a sesamoid) between the bases of metatarsals 2 and 3. Centrale present, roughly the size of T2+3. Prehallux subtriangular. Phalangeal formula reduced (1-2-3-4-3). Terminal phalanges of toes 2–4 with almost T-shaped knobs. Distribution, natural history, and conservation status. This species is known only from a single specimen from Andohahela National Park in southeast Madagascar ( Fig 6 ). The species is larger than the other members of the genus Mini , and its ecology may differ accordingly. Advertisement calls were not recorded. At present it is not possible to estimate its distribution or population status, and we prefer to suggest this species be considered Data Deficient until more information is available. Etymology. We use the specific epithet ‘ature’ as an arbitrary combination of letters, in order to form a pun on ‘miniature’ from the name in apposition, in reference to the small size of this species. It is to be regarded as an invariable noun.